μ-Opioid Inhibition of Ca 2+ Currents and Secretion in Isolated Terminals of the Neurohypophysis Occurs via Ryanodine-Sensitive Ca 2+ Stores

• Published in: The Journal of neuroscience 2014-03, Vol.34 (10), p.3733-3742
• Main Authors: Velázquez-Marrero, Cristina, Ortiz-Miranda, Sonia, Marrero, Héctor G., Custer, Edward E., Treistman, Steven N., Lemos, José R.
• Format: Article
• Language: English
• ISSN: 0270-6474; 1529-2401
• DOI: 10.1523/JNEUROSCI.2505-13.2014

μ-Opioid agonists have no effect on calcium currents ( I Ca ) in neurohypophysial terminals when recorded using the classic whole-cell patch-clamp configuration. However, μ-opioid receptor (MOR)-mediated inhibition of I Ca is reliably demonstrated using the perforated-patch configuration. This suggests that the MOR-signaling pathway is sensitive to intraterminal dialysis and is therefore mediated by a readily diffusible second messenger. Using the perforated patch-clamp technique and ratio-calcium-imaging methods, we describe a diffusible second messenger pathway stimulated by the MOR that inhibits voltage-gated calcium channels in isolated terminals from the rat neurohypophysis (NH). Our results show a rise in basal intracellular calcium ([Ca 2+ ] i ) in response to application of [ d -Ala 2 -N-Me-Phe 4 ,Gly5-ol]-Enkephalin (DAMGO), a MOR agonist, that is blocked by d -Phe-Cys-Tyr- d -Trp-Orn-Thr-Pen-Thr-NH 2 (CTOP), a MOR antagonist. Buffering DAMGO-induced changes in [Ca 2+ ] i with BAPTA-AM completely blocked the inhibition of both I Ca and high-K + -induced rises in [Ca 2+ ] i due to MOR activation, but had no effect on κ-opioid receptor (KOR)-mediated inhibition. Given the presence of ryanodine-sensitive stores in isolated terminals, we tested 8-bromo-cyclic adenosine diphosphate ribose (8Br-cADPr), a competitive inhibitor of cyclic ADP-ribose (cADPr) signaling that partially relieves DAMGO inhibition of I Ca and completely relieves MOR-mediated inhibition of high-K + -induced and DAMGO-induced rises in [Ca 2+ ] i . Furthermore, antagonist concentrations of ryanodine completely blocked MOR-induced increases in [Ca 2+ ] i and inhibition of I Ca and high-K + -induced rises in [Ca 2+ ] i while not affecting KOR-mediated inhibition. Antagonist concentrations of ryanodine also blocked MOR-mediated inhibition of electrically-evoked increases in capacitance. These results strongly suggest that a key diffusible second messenger mediating the MOR-signaling pathway in NH terminals is [Ca 2+ ] i released by cADPr from ryanodine-sensitive stores.